A critical early step in drug discovery and development is understanding the likely pharmacokinetics in the human body and the potential for multi-drug resistance. Early screening typically involves assays such as the parallel artificia membrane permeability assay (PAMPA) or the ATPase assay, which are simple to perform yet are not good predictors for drug transport in the body because of the crude description of the bilayers for the PAMPA assay and potential for false-negative and false-positive results in the ATPase assay. For promising drug candidates, more sophisticated cell-based assays using Caco-2 or Madin-Darby canine kidney (MDCK) cells are performed. However, these assays are much more costly and time-consuming to perform, and the value of the results is limited by the accuracy of the initial PAMPA and ATPase screens. We propose alternate assay strategies based on using droplet bilayers. These assays can provide results similar to the Caco-2 or MDCK assays with the lower complexity of the PAMPA or ATPase assays and higher throughput. Furthermore, these assays avoid long periods for cell growth, use very low quantities of transport protein and substrate, allow study of multiple transport protein types, and avoid issues associated with gaps in confluence of cells found in the Caco-2 or MDCK assays. Finally, the assays will allow ready comparison of active substrate transport and simple diffusion. This project is a collaboration between Drs. Faris and Dixit of SRI International (SRI), who developed the direct-deposition and laser-mediated droplet bilayer methods, and Dr. Gerk of Virginia Commonwealth University (VCU), who is an expert on transporter activity assays for drug development. To understand how the droplet bilayer assays conditions and results translate to conventional assays, to facilitate troubleshooting of the droplet assays, confirm reliability of the reagents, and transfer useful knowledge between SRI and VCU, comparable assay types will be performed at both SRI and VCU for the same two transporters and same substrates, and each group will visit the other while the assays are being performed. Flexible and multi-purpose assays for the activity of transporter proteins will improve our knowledge of basic biology and human health. To facilitate and accelerate the drug discovery and development processes, more sensitive, specific, faster, and cheaper methods are desired. We have a novel microdroplet technology capable of sensitive and accurate measurement of transport. Applying this technology to transporter proteins such as P-glycoprotein and Organic Anion Transporting Polypeptides would result in lowering research costs and increasing the success rate of new drug candidates.